A team of 17 researchers at the J. Craig Venter Institute (JCVI) has created the largest man-made DNA structure by synthesizing and assembling the 582,970 base pair genome of a bacterium, Mycoplasma genitalium JCVI-1.0. This work, published online today in the journal Science by Dan Gibson, Ph.D., et al, is the second of three key steps toward the team’s goal of creating a fully synthetic organism. In the next step, which is ongoing at the JCVI, the team will attempt to create a living bacterial cell based entirely on the synthetically made genome.
M. genitalium: Living Organism with the Smallest Genome.
The team achieved this technical feat by chemically making DNA fragments in the lab and developing new methods for the assembly and reproduction of the DNA segments. After several years of work perfecting chemical assembly, the team found they could use homologous recombination (a process that cells use to repair damage to their chromosomes) in the yeast Saccharomyces cerevisiae to rapidly build the entire bacterial chromosome from large subassemblies.
“This extraordinary accomplishment is a technological marvel that was only made possible because of the unique and accomplished JCVI team,” said J. Craig Venter, Ph.D., President and Founder of JCVI.
“Ham Smith, Clyde Hutchison, Dan Gibson, Gwyn Benders, and the others on this team dedicated the last several years to designing and perfecting new methods and techniques that we believe will become widely used to advance the field of synthetic genomics.”
The building blocks of DNA — adenine (A), guanine (G), cytosine (C) and thiamine (T) — are not easy chemicals to artificially synthesize into chromosomes. As the strands of DNA get longer they get increasingly brittle, making them more difficult to work with. Prior to today’s publication the largest synthesized DNA contained only 32,000 base pairs. Thus, building a synthetic version of the genome of the bacteria M. genitalium genome that has more than 580,000 base pairs presented a formidable challenge. However, the JCVI team has expertise in many technical areas and a keen biological understanding of several species of mycoplasmas.
“When we started this work several years ago, we knew it was going to be difficult because we were treading into unknown territory,” said Hamilton Smith, M.D., senior author on the publication.
“Through dedicated teamwork we have shown that building large genomes is now feasible and scalable so that important applications such as biofuels can be developed.”
Methods for Creating the Synthetic M. genitalium
The process to synthesize and assemble the synthetic version of the M. genitalium chromosome began first by resequencing the native M. genitalium genome to ensure that the team was starting with an error free sequence. After obtaining this correct version of the native genome, the team specially designed fragments of chemically synthesized DNA to build 101 “cassettes” of 5,000 to 7,000 base pairs of genetic code. As a measure to differentiate the synthetic genome versus the native genome, the team created “watermarks” in the synthetic genome. These are short inserted or substituted sequences that encode information not typically found in nature. Other changes the team made to the synthetic genome included disrupting a gene to block infectivity. To obtain the cassettes the JCVI team worked primarily with the DNA synthesis company Blue Heron Technology, as well as DNA 2.0 and GENEART.
Circular map of the M. genitalium chromosome. Genes are coloured according to the functional classification of the encoded proteins. The length of each projection is proportional to the number of amino acid residues in the respective protein. The outside ring shows the products of the genes that are transcribed clockwise, and the inside ring shows those transcribed counter clockwise. From here, the team devised a five stage assembly process where the cassettes were joined together in subassemblies to make larger and larger pieces that would eventually be combined to build the whole synthetic M. genitalium genome. In the first step, sets of four cassettes were joined to create 25 subassemblies, each about 24,000 base pairs (24kb). These 24kb fragments were cloned into the bacterium Escherichia coli to produce sufficient DNA for the next steps, and for DNA sequence validation. The next step involved combining three 24kb fragments together to create 8 assembled blocks, each about 72,000 base pairs. These 1/8th fragments of the whole genome were again cloned into E. coli for DNA production and DNA sequencing. Step three involved combining two 1/8th fragments together to produce large fragments approximately 144,000 base pairs or 1/4th of the whole genome. At this stage the team could not obtain half genome clones in E. coli, so the team experimented with yeast and found that it tolerated the large foreign DNA molecules well, and that they were able to assemble the fragments together by homologous recombination. This process was used to assemble the last cassettes, from 1/4 genome fragments to the final genome of more than 580,000 base pairs. The final chromosome was again sequenced in order to validate the complete accurate chemical structure. The synthetic M. genitalium has a molecular weight of 360,110 kilodaltons (kDa). Printed in 10 point font, the letters of the M. genitalium JCVI-1.0 genome span 147 pages.
This handout photo from the J. Craig Venter Institute shows a single molecule from the synthetic Mycoplasma genitalium bacteria over a period of 0.6 seconds. US scientists have taken a major step toward creating the first ever artificial life form by synthetically reproducing the DNA of a bacteria.
“This is an exciting advance for our team and the field. However, we continue to work toward the ultimate goal of inserting the synthetic chromosome into a cell and booting it up to create the first synthetic organism,” said Dan Gibson, lead author. The research to create the synthetic M. genitalium JCVI-1.0 was funded by Synthetic Genomics, Inc. Key Milestones in JCVI’s Synthetic Genomics Research The work described by Gibson et al. has its genesis in research by Dr. Venter and colleagues in the mid-1990s after sequencing M. genitalium and beginning work on the minimal genome project. This area of research, trying to understand the minimal genetic components necessary to sustain life, began with M. genitalium because it is a bacterium with the smallest genome that we know of that can be grown in pure culture. That work was published in the journal Science in 1995. In 2003 Drs. Venter, Smith and Hutchison made the first significant strides in the development of a synthetic genome by their work in assembling the 5,386 base pair bacteriophage ΦX174 (phi X). They did so using short, single strands of synthetically produced, commercially available DNA (known as oligonucleotides) and using an adaptation of polymerase chain reaction (PCR), known as polymerase cycle assembly (PCA), to build the phi X genome. The team produced the synthetic phi X in just 14 days. In June 2007 another major advance was achieved when JCVI researchers led by Carole Lartigue, Ph.D., announced the results of work on genome transplantation methods allowing them to transform one type of bacteria into another type dictated by the transplanted chromosome. The work was published in the journal Science, and outlined the methods and techniques used to change one bacterial species, Mycoplasma capricolum, into another, Mycoplasma mycoides Large Colony (LC), by replacing one organism’s genome with the other one’s genome. Genome transplantation was the first essential enabling step in the field of synthetic genomics as it is a key mechanism by which chemically synthesized chromosomes can be activated into viable living cells. Today’s announcement of the successful synthesis of the M. genitalium genome is the second step leading to the next experiments to transplant a fully synthetic bacterial chromosome into a living organism and “boot up” the cell. Ethical Considerations Since the beginning of the quest to understand and build a synthetic genome, Dr. Venter and his team have been concerned with the societal issues surrounding the work. In 1995 while the team was doing the research on the minimal genome, the work underwent significant ethical review by a panel of experts at the University of Pennsylvania (Cho et al, Science December 1999:Vol. 286. no. 5447, pp. 2087 – 2090). The bioethical group's independent deliberations, published at the same time as the scientific minimal genome research, resulted in a unanimous decision that there were no strong ethical reasons why the work should not continue as long as the scientists involved continued to engage public discussion. Dr. Venter and the team at JCVI continue to work with bioethicists, outside policy groups, legislative members and staff, and the public to encourage discussion and understanding about the societal implications of their work and the field of synthetic genomics generally. As such, the JCVI’s policy team, along with the Center for Strategic & International Studies (CSIS), and the Massachusetts Institute of Technology (MIT), were funded by a grant from the Alfred P. Sloan Foundation for a 20-month study that explored the risks and benefits of this emerging technology, as well as possible safeguards to prevent abuse, including bioterrorism. After several workshops and public sessions the group published a report in October 2007 outlining options for the field and its researchers. About the J. Craig Venter Institute The JCVI is a not-for-profit research institute dedicated to the advancement of the science of genomics; the understanding of its implications for society; and communication of those results to the scientific community, the public, and policymakers. Founded by J. Craig Venter, Ph.D., the JCVI is home to approximately 400 scientists and staff with expertise in human and evolutionary biology, genetics, bioinformatics/informatics, information technology, high-throughput DNA sequencing, genomic and environmental policy research, and public education in science and science policy. The legacy organizations of the JCVI are: The Institute for Genomic Research (TIGR), The Center for the Advancement of Genomics (TCAG), the Institute for Biological Energy Alternatives (IBEA), the Joint Technology Center (JTC), and the J. Craig Venter Science Foundation. The JCVI is a 501 (c)(3) organization. Publication: Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome Published Online January 24, 2008Science DOI: 10.1126/science.1151721
Links: J. Craig Venter Institute Synthetic biology for energy production Mycoplasma genitalium genome More from CellNEWS: Venter makes synthetic life... or not yet? October 09, 2007 Next Step Towards Artificial Life Whole Genome Transplantation Achieved in Mycobacterium June 28, 2007 Venter attempt at the minimalistic approach of creating ‘artificially-made’ life November24, 2002 ......... ZenMaster
For more on stem cells and cloning, go to CellNEWS at http://www.geocities.com/giantfideli/index.html
Posts
No comments:
Post a Comment